Phorbol ester compositions and methods of use for treating or reducing the duration of cytopenia

ABSTRACT

Methods and compositions containing a phorbol ester or a derivative of a phorbol ester in combination with G-CSF or in combination with EPO, are provided for the treatment of cytopenia in mammalian subjects. The compositions and methods also reduce the duration of cytopenia such as neutropenia, thrombocytopenia, and/or anemia.

TECHNICAL FIELD

The present invention relates to the use of phorbol esters for thetreatment of cytopenia. Specifically, the present invention relates tothe use of phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate(TPA) or phorbol-12-myristate (PMA), and G-CSF in the treatment andreduction of neutropenia and thrombocytopenia in patients with aneoplastic disease. The present invention also relates to the use ofphorbol esters, such as TPA and erythropoeitin (EPO) for the treatmentof anemia in patients.

BACKGROUND

Plants have historically served many medicinal purposes. The WorldHealth Organization (WHO) estimates that 4 billion people, 80% of theworld population, presently use herbal medicine for some aspect ofprimary health care. (WHO Fact sheet Fact sheet No 134 December 2008)However, it can be difficult to isolate the specific compound that hasthe medicinal effect and to reproduce it on a commercial scale.Additionally, while active compounds may be isolated from a plant, theother parts of a plant such as the minerals, vitamins, volatile oils,glycosides, alkaloids, bioflavanoids, and other substances may also beinvolved in the functioning of the active ingredient or the medicinaleffect for which the plant is known, making the use, purification andcommercialization of plant based pharmaceutical agents a challenge.

Phorbol is a natural, plant-derived organic compound of the tiglianefamily of diterpenes. It was first isolated in 1934 as a hydrolysisproduct of croton oil derived from the seeds of Croton tiglium, a leafyshrub of the Euphorbiaceae family that is native to Southeastern Asia.Various esters of phorbol have important biological properties includingthe reported ability to mimic diacylglycerols and activate proteinkinase C (PKC); and to modulate downstream cell signaling pathwaysincluding the mitogen-activated protein kinase (MAPK) pathways. Phorbolesters are additionally thought to bind to chimaerins, the Ras activatorRasGRP, and the vesicle-priming protein Munc-13 (Brose N, Rosenmund C.,J Cell Sci; 115:4399-411 (2002)). Some phorbol esters also inducenuclear factor-kappa B (NF-κB). The most notable physiological propertyof phorbol esters is their reported capacity to act as tumor promoters.(Blumberg, 1988; Goel, G et al., Int, Journal of Toxicology 26, 279-288(2007)).

12-O-tetradecanoylphorbol-13-acetate (TPA), also calledphorbol-12-myristate-13-acetate (PMA), is a phorbol ester used in modelsof carcinogenesis as an inducer for differentiation and/or apoptosis inmultiple cell lines and primary cells. TPA has also been reported tocause an increase in circulating white blood cells and neutrophils inpatients whose bone marrow function has been depressed by chemotherapy(Han Z. T. et al. Proc. Natl. Acad. Sci. 95, 5363-5365 (1998)). However,due to a variety of factors, including caustic reactions when contactedwith the skin and concerns for its potential toxicity, TPA has not beenshown to be an effective adjuvant to chemotherapy. Indeed, as phorbolesters play a key role in activation of protein kinase C, which triggersvarious cellular responses resulting in inflammatory responses and tumordevelopment (Goel et al., Int, Journal of Toxicology 26, 279-288(2007)), phorbol esters would generally be excluded from possibletreatment candidates for neoplasms including cancer.

Cancer is one of the leading causes of death worldwide accounting for7.6 million deaths (around 13% of all deaths) in 2008 (GLOBOCAN 2008(IARC) (Section of Cancer Information (Aug. 12, 2011)). Globally,12,662,600 new cases were diagnosed in 2008. (2008 (GLOBOCAN 2008(IARC). In the U.S. alone, 1,596,670 new cases of cancer were diagnosedin 2011 (Cancer Facts & Figures—2011, American Cancer Society (ACS),Atlanta, Ga., 2011).

Cancer treatments generally involve a combination of surgery,chemotherapy, hormonal therapy and/or radiation treatment to eradicateneoplastic cells in a patient. However, current therapeutics forneoplasms have a number of drawbacks including insufficient potency andintolerable side effects. Surgery, for example, may be contraindicateddue to the health of a patient. Additionally, it may be difficult toobtain clear margins around a tumor, resulting in some neoplastic tissuebeing left behind and an increased chance of recurrence of the disease.

Generally, chemotherapeutics act by killing cells that divide rapidly,one of the main properties of most cancer cells. However, they also harmnormal cells that divide rapidly such as cells in bone marrow, thedigestive tract and hair follicles. They frequently have significantside effects including severe nausea, bone marrow depression, andimmunosuppression.

Ionizing radiation works by damaging the DNA of exposed tissue. However,while targeted, it can still damage normal cells as well as neoplasmsand can have side effects such as anemia, nausea and vomiting, poorappetite, weight loss, constipation, diarrhea, hair loss, andinfertility.

For many patients, the toxic side effects of current therapies diminishtheir quality of life to such an extent they simply stop taking theirmedications. For others, therapeutic schedules are so complicated andinconvenient that compliance is limited. Other patients experienceexcellent results initially, but suffer relapses despite full compliancewith therapeutic regimens. There is clearly a need for new and moreeffective treatments for neoplasms and to manage the side effects ofcurrent treatments for neoplasms including cancer.

SUMMARY OF THE INVENTION

The present invention relates to compositions containing and methods ofusing phorbol esters of Formula I in combination with G-CSF. Thecompositions and methods described herein are effective in treating andreducing the duration of neutropenia and thrombocytopenia in patientswith neoplastic conditions.

In an embodiment, the present invention relates to a method of treatingcytopenia comprising administering to a mammalian subject in needthereof, a phorbol ester of Formula I (as described herein),pharmaceutically-acceptable salt, isomer, enantiomer, solvate, hydrate,polymorph or prodrug thereof, wherein R₁ and R₂ are selected from thegroup consisting of hydrogen, hydroxyl,

and substituted derivatives thereof, R₃ is selected from hydrogen,

and substituted derivatives thereof;in combination with a granulocyte-colony stimulating factor (G-CSF).

In a particular embodiment, the present invention relates to a a methodof treating neutropenia and/or thrombocytopenia comprising administeringto a mammalian subject in need thereof, a combination of a phorbol esterof Formula I, pharmaceutically-acceptable salt, isomer, enantiomer,solvate, hydrate, polymorph or prodrug thereof; in combination with agranulocyte-colony stimulating factor (G-CSF).

In another embodiment, the present invention relates to a method oftreating cytopenia comprising administering to a mammalian subject inneed thereof, a phorbol ester of Formula I, pharmaceutically-acceptablesalt, isomer, enantiomer, solvate, hydrate, polymorph or prodrugthereof; in combination with an erythropoietin (EPO).

In a particular embodiment, the present invention relates to a a methodof treating anemia comprising administering to a mammalian subject inneed thereof, a phorbol ester of Formula I, pharmaceutically-acceptablesalt, isomer, enantiomer, solvate, hydrate, polymorph or prodrugthereof; in combination with an erythropoietin (EPO).

In methods of the present invention, R₁ or R₂ of Formula I is

the remaining R₁ or R₂ is

and R₃ of Formula I is hydrogen.

In particular, in the methods of the invention, the phorbol ester isphorbol 13-butyrate, phorbol 12-decanoate, phorbol 13-decanoate, phorbol12,13-diacetate, phorbol 13,20-diacetate, phorbol 12,13-dibenzoate,phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate, phorbol12,13-dihexanoate, phorbol 12,13-dipropionate, phorbol 12-myristate,phorbol 13-myristate, phorbol 12,13,20-triacetate, 12-deoxyphorbol13-angelate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate,12-deoxyphorbol 13-phenylacetate, 12-deoxyphorbol 13-phenylacetate20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tigliate13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate, or phorbol13-acetate.

In a preferred embodiment, the phorbol ester is12-O-tetradecanoylphorbol-13-acetate (TPA).

The methods of the present invention, may further comprise administeringat least one secondary or adjunctive therapeutic agent.

In certain embodiments of the present invention, G-CSF is administeredto said subject in a coordinate administration protocol, simultaneouslywith, prior to, or after, administration of said phorbol ester ofFormula I.

In certain embodiments of the present invention, EPO is administered tosaid subject in a coordinate administration protocol, simultaneouslywith, prior to, or after, administration of said phorbol ester ofFormula I.

The methods of the present invention involve administering the phorbolester of Formula I in an effective amount comprising between about 10and 1500 μg of said phorbol ester of Formula I every day or every otherday.

In certain embodiments, the methods of the present invention involveadministering the phorbol ester of Formula I in an effective amountcomprising between about 150 to 500 μg of said phorbol ester orderivative compound of Formula I every day or every other day.

In preferred embodiment of the present invention, the combination of thephorbol ester of Formula I and G-CSF increases absolute neutrophil count(ANC) of the mammalian subject to above 1500/mm³.

In another preferred embodiment, the combination of the phorbol ester ofFormula I and G-CSF increases platelet levels of the mammalian subjectto above 100,000/μl.

In a certain preferred embodiment of the present invention, thecombination of the phorbol ester of Formula I and EPO increases acomplete blood count (CBC) level measured in a complete blood count byat least 10%.

In another preferred embodiment, wherein the combination of the phorbolester of Formula I and EPO increases a hemoglobin level of the mammaliansubject to above a normal hemoglobin level.

In a preferred embodiment, the methods of the present invention involvetreating or reducing cytopenia such as neutropenia, thrombocytopeniaand/or anemia, in a human with acute myeloid leukemia (AML).

In another embodiment, the present invention relates to compositionscontaining a phorbol ester of Formula I and G-CSF.

In a preferred embodiment, the phorbol ester of Formula I is present inan effective amount sufficient to treat or reduce the duration ofcytopenia, such as neutropenia and/or thrombocytopenia.

In a preferred embodiment, the compositions of the present inventioncontain TPA as the phorbol ester, and the TPA and G-CSF are present inan effective amount to treat or reduce the duration of cytopenia, suchas neutropenia and/or thrombocytopenia. In a particularly preferredembodiment, the effective amount may be a synergistically effectiveamount to treat or reduce the duration of neutropenia and/orthrombocytopenia.

The present invention also relates to compositions containing a phorbolester of Formula I and EPO.

In a preferred embodiment, the phorbol ester of Formula I is present inan effective amount sufficient to treat or reduce the duration ofcytopenia, such as anemia.

In a preferred embodiment, the compositions of the present inventioncontain TPA as the phorbol ester, and the TPA and EPO are present in aneffective amount to treat or reduce the duration of cytopenia, such asanemia. In a particularly preferred embodiment, the effective amount maybe a synergistically effective amount to treat or reduce the duration ofanemia.

In another embodiment, the neutropenia, thrombocytopenia and/or anemiais related to treatment of neoplasms. Such neoplasms may be malignant orbenign. In some embodiments, neoplasms may be solid or non-solidcancers. In other embodiments, the neoplasms may be relapses. In anotherembodiment, the neoplasms may be refractory.

Exemplary neoplasms include, but are not limited to, hematologicmalignancies/bone marrow disorders, including, but not limited to,leukemia, including acute myeloid leukemia (AML), chronic myeloidleukemia (CML), chronic myeloid leukemia blast crisis, myelodysplasia,and myeloproliferative syndrome; lymphoma, including Hodgkin's andnon-Hodgkin's lymphoma; subcutaneous adenocarcinoma; ovarianteratocarcinoma; liver cancer; breast cancer; bone cancer; lung cancer;pancreatic cancer; non-small cell lung cancer; and prostate cancer.Other neoplastic conditions amenable to treatment using the methods andcompositions as described herein include other cancer disorders andconditions, including solid tumors of various types.

In yet another embodiment, the phorbol esters and derivatives of phorbolesters as described herein may be used to modulate cell signalingpathways. Such modulation may have a variety of results, for example, insome embodiments, the use of compositions containing phorbol esters andderivatives of phorbol esters may increase white blood cell counts inmammalian subjects. In another embodiment, compositions containingphorbol esters and/or phorbol ester derivatives may alter the release ofTh1 cytokines in mammalian subjects. In a further embodiment,compositions containing phorbol esters and/or phorbol ester derivativesmay alter the release of interleukin 2 (IL-2) in mammalian subjects. Inan additional embodiment, compositions containing phorbol esters and/orphorbol ester derivatives may alter the release of interferon inmammalian subjects. In yet another embodiment, compositions containingphorbol esters and/or phorbol ester derivatives may alter the rate ofERK phosphorylation.

The invention achieves the foregoing and satisfies additional objectsand advantages by providing novel and surprisingly unexpected methodsand compositions useful for treating or reducing the duration ofcytopenia, such as neutropenia, thrombocytopenia, and anemia.

DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates the synergistic effect achieved by the combination ofTPA and GCSF. TPA stimulates upstream stem cells to differentiate intodownstream stem cells, while GCSF stimulates the downstream stem cells.TPA also stimulates downstream stem cells. Neutrophils are one type ofgranulocyte.

FIG. 2 illustrates the combination of TPA and GCSF generates strongerstimulating effects than TPA or G-CSF alone. The below abbreviations areused in FIG. 2 and throughout the present disclosure with respect to thefollowing terminology.

-   CFCs colony-forming cells-   CFU-E colony forming unit-erythroid-   CFU-G colony forming unit-granulocyte-   CFU-GEMM colony forming unit-granulocyte, erythrocyte, macrophage,    megakaryocyte-   CFU-GM colony forming unit-granulocyte, macrophage-   CFU-M colony forming unit-macrophage-   BFU-E Burst forming unit erythroid

The forgoing and additional objects, features, aspects and advantages ofthe present invention will become apparent from the following detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION Definitions

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are collected here. These definitions should be read in light ofthe remainder of the disclosure and understood as by a person of skillin the art. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as would be understood by a person ofordinary skill in the art.

“G-CSF” or “GCSF” is known as granulocyte-colony stimulating factor orcolony-stimulating factor 3 (“CSF3”), and may be used interchangeablyherein. “G-CSF,” “GCSF,” or “CSF3” is a glycoprotein that stimulates thebone marrow to produce granulocytes and stem cells and release them intothe bloodstream.

“EPO” is known as erythropoietin, which is a glycoprotein hormone thatcontrols erythropoiesis, or red blood cell production.

Induction therapy is used herein to mean the first phase of treatmentfor a disease, typically, cancer. For example, the goal of inductiontherapy for acute myeloid leukemia is to produce a complete remission inthe bone marrow and return to normal blood counts.

Consolidation therapy is used herein to mean treatment(s) given aftercancer has disappeared following initial treatment, and is given toprevent recurrence of cancer. Consolidation therapy is used to kill anycancer cells that may be left in the body.

Cytopenia is used herein to mean a reduction in the number of bloodcells, and includes low red blood cell count (anemia), low white bloodcell count (leukopenia or neutropenia), low platelet count(thrombocytopenia), or a combination thereof (pancytopenia), and lowgranulocyte count (granulocytopenia).

Red blood cell count (RBC) is the number of red blood cells capable ofcarrying hemoglobin in a mm³ of blood. The normal RBC for men is 4.5 to6 million mm³; for women, 4 to 5.5 million per mm³. Seecytopenia-cancertype.blogspot.ca/2007/12/diagnosis-of-cytopenia.html.

White blood cell count (WBC) is the total number of all five types ofwhite blood cells. The normal WBC for men and women is 5,000 to 10,000per mm of blood. Seecytopenia-cancertype.blogspot.ca/2007/12/diagnosis-of-cytopenia.html.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The term “consisting essentially of” is used to limit the elements tothose specified and those that do not materially affect the basic andnovel characteristics of the material or steps.

The term “including” is used herein to mean “including but not limitedto.” “Including” and “including but not limited to” are usedinterchangeably.

A “patient,” “subject” or “host” to be treated by the subject method maymean either a mammal such as a human, or non-human mammal.

The term “pharmaceutically-acceptable carrier” is an art-recognized termand refers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material. Such carrier must be “acceptable” in thesense of being compatible with the subject composition and itscomponents and not injurious to the patient. Examples include, but arenot limited to, binders, fillers, lubricants, emulsifiers, suspendingagents, sweeteners, flavorings, preservatives, buffers, wetting agents,disintegrants, effervescent agents and other conventional excipients andadditives.

The term “treating” is an art-recognized term and refers to curing aswell as ameliorating or reducing at least one symptom of any conditionor disorder.

The term “therapeutic agent” or “drug” is an art-recognized and refersto any chemical moiety that is a biologically, physiologically, orpharmacologically active substance that acts locally or systemically ina subject. For example, therapeutic agents or drugs, are described inthe Merck Index, the Physicians' Desk Reference, and The PharmacologicalBasis of Therapeutics.

The term “effective amount” is therapeutically effective, in single ormultiple unit dosage form. The effective amount is an amount that issufficient to provide a therapeutic effect in a mammal, including ahuman. For example, an effective amount may be an amount sufficient tomeasurably treat or reduce/shorten the duration of neutropenia and/orthrombocytopenia in a subject. Another example of an effective amount isan amount sufficient to measurably treat or reduce/shorten the durationof anemia. Dosage levels or amounts of the particular therapeutic agentor drug used to provide a therapeutically effective amount varydepending on factors including, but not limited to, age, weight, gender,medical condition of the mammal/human, and the route of administration.Effective amounts of a phorbol ester compound or related or derivativecompound of Formula I (e.g., a unit dose comprising an effectiveconcentration/amount of TPA, or of a selected pharmaceuticallyacceptable salt, isomer, enantiomer, solvate, polymorph and/or prodrugof TPA), of G-CSF, or of EPO, will be readily determined by those ofordinary skill in the art, depending on clinical and patient-specificfactors. A therapeutically effective amount according to the presentinvention may include a synergistically effective amount.

Cytopenia has traditionally been classified as a deficiency related(i.e., nutritional or hormonal deficiency), immune mediated, BM failurebased, or idiopathic cytopenias. See Valent, P., Hematology: 485-491(2012), the disclosure of which is herein incorporated by reference inits entirety.

Diagnosis of cytopenia in a cancer patient requires a complete bloodcount (CBC) and the identification of any blood and bone marrowabnormalities, such as anemia, neutropenia, or thrombocytopenia. Seecytopenia-cancertype.blogspot.ca/2007/12/diagnosis-of-cytopenia.html.

Chemotherapeutic agents adversely affect bone marrow cells, and acomplete blood count (CBC) is necessary prior to each treatment. Theeffects on bone marrow are temporary and normal functioning usuallyreturns within 4-10 days, but white blood cells have a life span of 1 to3 days; so although those WBCs in circulation remain unaffected, theslow production of new leukocytes creates a period of increased risk forinfection. Seecytopenia-cancertype.blogspot.ca/2007/12/diagnosis-of-cytopenia.html. Ifwhite blood cell production does not recover before the next treatment,treatment is delayed until the cell count increases sufficiently. Id.Mature red blood cells have a relatively long life (120 days), cellproduction usually resumes before symptoms of deficiency develop. Id.

Anemia is a deficiency in erythrocytes that reduces the amount of oxygenreaching all cells in the body, so that all tissue and organ function isimpaired. Anemia produces symptoms including severe fatigue, confusion,dizziness, headache, lightheadedness, loss of concentration, pallor(pale skin, nail beds, gums, linings of eyelids), rapid heart rate(tachycardia), and shortness of breath (dyspnea). Seecytopenia-cancertype.blogspot.ca/2007/12/cytopenia-signs-and-symptoms.html.Individuals with anemia are advised to rest and eat foods high in iron,and treatment may include medication that helps restore the red bloodsupply (such as erythropoietin) and a transfusion of packed red bloodcells. Seecytopenia-cancertype.blogspot.ca/2007/12/cytopenia-treatment.html. TheFood and Drug Administration (FDA) in March 2007 issued a warning aboutthese medications in response to studies indicating that they mayincrease the risk for blood clots, strokes, and heart attacks in somepatients (e.g., patients who have kidney disease). Id.

Neutropenia is a white blood cell deficiency with symptoms includingfrequent and/or severe bacterial, viral, and/or fungal infections;fever; and mouth and throat ulcers. Seecytopenia-cancertype.blogspot.ca/2007/12/cytopenia-signs-and-symptoms.html.A colony-stimulating factor (CSF), may be prescribed to speed thedevelopment of white blood cells and shorten the period ofsusceptibility to infection. Seecytopenia-cancertype.blogspot.ca/2007/12/cytopenia-treatment.html.

Thrombocytopenia is a platelet deficiency that causes patients to bruiseand bleed easily, and is characterized by symptoms including bleeding nthe mucous membranes lining the mouth, nose, colon, and vagina. Seecytopenia-cancertype.blogspot.ca/2007/12/cytopenia-signs-and-symptoms.html.It is characterized by a below normal platelet count of 15,000 to300,000 per milliliter and the risk of increased bleeding usually peaks10 to 14 days following a course of chemotherapy. Id. A persistentlydecreased platelet count may be treated with a transfusion of platelets.See cytopenia-cancertype.blogspot.ca/2007/12/cytopenia-treatment.html.

Growth factors (such as Epoetin alpha (Procrit®, Epogen®), G-CSF(granulocyte colony-stimulating factor; e.g., filgrastim [Neupogen®),and GM-CSF (granulocyte-macrophage colony-stimulating factor)) aresynthetic versions of substances involved in stimulating red and whiteblood cell production, but caution is exercised when prescribing thesemedications for people with tumors that involve the bone marrow, becausegrowth factors might stimulate malignant cell growth. Seecytopenia-cancertype.blogspot.ca/2007/12/cytopenia-treatment.html. Theside effects associated with these growth factors include fever,fatigue, dizziness, diarrhea, nausea, vomiting, weakness, andparesthesia (prickling sensation) (with epoetin alpha); and bone painwith (G-CSF). Id.

Chemotherapy and radiation therapy both reduce the number ofblood-forming stem cells in cancer patients, but chemotherapeutic agentshave a greater adverse effect because they suppress bone marrow functionin several ways—the extent of damage is related to the particulardrug(s) and the dose used. Seecytopenia-cancertype.blogspot.ca/2007/12/cytopenia-causes-and-risk-factors.html.

Deficiencies in blood cell types can be caused by chemotherapeuticagents which damage blood-forming stem cells, suppress the kidneys'production of erythropoietin (hormone that stimulates blood cellproduction), and trigger red cell destruction (hemolysis) by inducing animmune response that causes the body to mistakenly identify erythrocytesas foreign bodies and destroy them. Seecytopenia-cancertype.blogspot.ca/2007/12/cytopenia-causes-and-risk-factors.html.However, anemia, thrombocytopenia, and neutropenia caused by cancertreatment are usually resolved once the course of treatment is over. Seecytopenia-cancertype.blogspot.ca/2007/12/cytopenia-treatment.html.

Malignant tumors can also cause anemia and other cytopenias when theydirectly invade bone marrow and suppress marrow function. Seecytopenia-cancertype.blogspot.ca/2007/12/cytopenia-causes-and-risk-factors.html.

The compositions and methods as described herein may be used to treat orreduce/shorten the duration of anemia, neutropenia and/orthrombocytopenia in mammalian subjects, including humans. In someembodiment, the mammalian subject is a human with neoplastic disease.

Compositions and methods of using a phorbol ester of Formula I, below:

wherein R₁ and R₂ may be hydrogen; hydroxyl;

wherein the alkyl group contains 1 to 15 carbon atoms;

wherein a lower alkenyl group contains between 1 to 7 carbon atoms;

and substituted derivatives thereof. R₃ may be hydrogen or

and substituted derivatives thereof; in combination with G-CSF fortreatment of cytopenia including but not limited to, neutropenia and/orthrombocytopenia. The methods and compositions of the present inventionfurther include any pharmaceutical salts, enantiomers, isomer,polymorphs, prodrugs, hydrates and solvates of the compositions ofFormula I; in combination with G-CSF for treatment of neutropenia and/orthrombocytopenia. For example, the combination of phorbol ester ofFormula I with G-CSF is also useful for reducing or shortening theduration of neutropenia and/or thrombocytopenia.

Compositions and methods of using a phorbol ester of Formula I, below:

wherein R₁ and R₂ may be hydrogen; hydroxyl;

wherein the alkyl group contains 1 to 15 carbon atoms;

wherein a lower alkenyl group contains between 1 to 7 carbon atoms;

and substituted derivatives thereof. R₃ may be hydrogen or

and substituted derivatives thereof; in combination with EPO fortreatment of cytopenia, including but not limited to, anemia. Themethods and compositions of the present invention further include anypharmaceutical salts, enantiomers, isomer, polymorphs, prodrugs,hydrates and solvates of the compositions of Formula I; in combinationwith EPO for treatment of anemia. For example, the combination ofphorbol ester of Formula I with EPO is also useful for reducing orshortening the duration of anemia.

In some embodiments, at least one of R₁ and R₂ are other than hydrogenand R₃ is hydrogen or

and substituted derivatives thereof. In another embodiment, either R₁ orR₂ is

the remaining R₁ or R₂ is a

wherein a lower alkyl is between 1 and 7 carbons, and R₃ is hydrogen.

The alkyl, alkenyl, phenyl and benzyl groups of the formulas herein maybe unsubstituted or substituted with halogens, preferably, chlorine,fluorine or bromine; nitro; amino; and/or similar type radicals.

Compositions and methods using the same include a combination of aphorbol ester of Formula II, as 12-O-tetradecanoylphorbol-13-acetate(TPA):

with G-CSF, for treatment of cytopenia, including but not limited to,neutropenia and/or thrombocytopenia. For example, the combination of TPAwith G-CSF is also useful for reducing or shortening the duration ofneutropenia and/or thrombocytopenia

Compositions and methods using the same include a combination of aphorbol ester of Formula II, as 12-O-tetradecanoylphorbol-13-acetate(TPA):

with EPO, for treating cytopenia, including but not limited to, anemia.For example, the combination of TPA with EPO is also useful for reducingor shortening the duration of anemia.

Useful phorbol esters of Formula I and related compounds and derivativeswithin the formulations and methods of the invention include, but arenot limited to, other pharmaceutically acceptable active salts of saidcompounds, as well as active isomers, enantiomers, polymorphs,glycosylated derivatives, solvates, hydrates, and/or prodrugs of saidcompounds. Exemplary forms of phorbol esters for use within thecompositions and methods of the invention include, but are not limitedto, phorbol 13-butyrate; phorbol 12-decanoate; phorbol 13-decanoate;phorbol 12,13-diacetate; phorbol 13,20-diacetate; phorbol12,13-dibenzoate; phorbol 12,13-dibutyrate; phorbol 12,13-didecanoate;phorbol 12,13-dihexanoate; phorbol 12,13-dipropionate; phorbol12-myristate; phorbol 13-myristate; phorbol 12-myristate-13-acetate(also known as TPA or PMA); phorbol 12,13,20-triacetate; 12-deoxyphorbol13-angelate; 12-deoxyphorbol 13-angelate 20-acetate; 12-deoxyphorbol13-isobutyrate; 12-deoxyphorbol 13-isobutyrate-20-acetate;12-deoxyphorbol 13-phenylacetate; 12-deoxyphorbol 13-phenylacetate20-acetate; 12-deoxyphorbol 13-tetradecanoate; phorbol 12-tigliate13-decanoate; 12-deoxyphorbol 13-acetate; phorbol 12-acetate; andphorbol 13-acetate.

A broad range of mammalian subjects, including human subjects, areamenable to treatment using the compositions and methods of theinvention. These subjects include, but are not limited to, individualssuffering from diseases or conditions including but not limited to,neoplastic diseases, side effects from chemotherapy, side effects fromradiation therapy, prostate hypertrophy, urinary incontinence,Myasthemia gravis, and kidney disease.

Mammalian subjects that are amenable to treatment with phorbol esters ofFormula I, or derivative of the phorbol esters of the Formula I,particularly TPA, in combination with GCSF or EPO according to themethods of the present invention include subjects suffering from anemia,neutropenia and/or thrombocytopenia. Such subjects amenable to treatmentwith phorbol esters of Formula I, particularly TPA, in combination withGCSF or EPO include those suffering from symptoms of diseases ordisorders including but not limited to, neoplastic diseases or effectscaused by treatment of the neoplastic disease.

Additional mammalian subjects, including humans, amenable to treatmentwith compositions and methods as described herein, particularly TPA,according to the methods of the present invention include subjects orindividuals with anemia related diseases or conditions, including butnot limited to, anemia related to kidney failure or disease, anemiarelated to pregnancy, anemia related to poor nutrition, perniciousanemia, sickle cell anemia, thalassemia, alcoholism, bone marrow-relatedanemia (such as leukemia or lymphoma), aplastic anemia (from viralinfections), anemia related to medications (such as cancer medications,HIV medications, seizure medications, transplant medications, malariamedications, antibiotics, antifungal, and antihistamines), hemolyticanemia, anemia related to thyroid problems, anemia related to liverdisease, and autoimmune disease (such as lupus).

Additional mammalian subjects, including humans, amenable to treatmentwith compositions and methods as described herein, particularly TPA,according to the methods of the present invention include subjects orindividuals with neutropenia related diseases or conditions, includingbut not limited to, congenital neutropenia (such as Kostmann'ssyndrome), cyclic neutropenia, idiopathic neutropenia, autoimmuneneutropenia, and drug-induced neutropenia (such as from cancer drugs).

Additional mammalian subjects, including humans, amenable to treatmentwith compositions and methods as described herein, particularly TPA,according to the methods of the present invention include subjects orindividuals with thrombocytopenia related diseases or conditions,including but not limited to, viral infections (such as parvovirus,rubella, mumps, varicella, hepatitis C, Epstein-Barr virus, and HIV),severe infections or sepsis, drug-induced thrombocytopenia (such as fromcancer drugs, thiazide, sulfonamide antibiotics, carbamazepine, digoxin,quinine, quinidine, acetaminophen, heparin, and ripampin), transfusionreactions, rheumatologic conditions (such as systemic lupuserythematosus), and idiopathic thrombocytopenia purpura. These and othersubjects are effectively treated prophylactically and/ortherapeutically, by administering to the subject an effective amount ofa phorbol ester of Formula I or derivative of a phorbol ester of FormulaI sufficient to treat and/or reduce the duration of anemia, neutropeniaand/or thrombocytopenia in mammalian subjects with a neoplastic disease.

Chemotherapy is the treatment of cancer with an anti-neoplastic drug orcombination of such drugs. Chemotherapy works by impairing thereproduction of rapidly splitting cells, a property common in cancerouscells. However it does not actively distinguish between healthy cellsthat are also rapidly splitting and cancerous cells and it has a numberof side effects such as, but not limited to, neutropenia, anemia, andthrombocytopenia.

Mammalian subjects amenable to treatment with phorbol esters of FormulaI, particularly TPA, according to the methods of the present inventionadditionally include, but are not limited to, mammalian subjectsundergoing chemotherapy.

Mammalian subjects suffering from neoplastic disease include malignantneoplastic diseases such as solid and non-solid cancers. Non-solidcancers may include, hematologic malignancies/bone marrow disorders,including, but not limited to, leukemia, including acute myeloidleukemia (AML), chronic myeloid leukemia (CML), chronic myeloid leukemiablast crisis, myelodysplasia, myeloproliferative syndrome. Solid cancersmay include, but are not limited to, lymphoma, including Hodgkin's andnon-Hodgkin's lymphoma, subcutaneous adenocarcinoma, ovarianteratocarcinoma, lung cancer; bone cancer; breast cancer; liver cancer;pancreatic cancer; oral cancer; non-small cell lung cancer and prostatecancer.

Therapeutically useful methods and formulations of the invention willeffectively use a phorbol ester of Formula I in a variety of forms, asnoted above, including any active, pharmaceutically acceptable salts ofsaid compounds, as well as active isomers, enantiomers, polymorphs,solvates, hydrates, prodrugs, and/or combinations thereof. TPA offormula II is employed as an illustrative embodiment of the inventionwithin the examples herein below.

Within additional aspects of the invention, combinatorial formulationsand methods are provided which employ an effective amount of a phorbolester of Formula I or derivative of a phorbol ester of Formula I incombination with one or more secondary or adjunctive active agent(s)that is/are combinatorially formulated or coordinately administered withthe phorbol ester compound of Formula I to yield an effective responsein the subject.

A phorbol ester compound of Formula I or derivative of the phorbol esterof Formula I is used in combination with G-CSF. Specifically, G-CSF isused in combination with a phorbol ester, e.g., TPA.

A phorbol ester compound of Formula I or derivative of the phorbol esterof Formula I is used in combination with erythropoeitin (EPO).Specifically, EPO is used in combination with TPA.

A phorbol ester compound of Formula I or derivative of the phorbol esterof Formula I is used in combination with G-CSF. Specifically, G-CSF isused in combination with TPA.

Compositions as described herein comprise G-CSF and a phorbol estercompound of Formula I or derivative compound of phorbol esters ofFormula I including pharmaceutically acceptable salts, enantiomers,isomers, polymorphs, prodrugs, hydrates and solvates thereof, in aneffective amount to treat or reduce the duration of neutropenia and/orthrombocytopenia.

Compositions as described herein comprise EPO and a phorbol estercompound of Formula I or derivative compound of phorbol esters ofFormula I including pharmaceutically acceptable salts, enantiomers,isomers, polymorphs, prodrugs, hydrates and solvates thereof, in aneffective amount to treat or reduce the duration of anemia.

The compositions of the invention comprise G-CSF and a phorbol estercompound of Formula I or derivative compound of phorbol esters ofFormula I including pharmaceutically acceptable salts, enantiomers,isomers, polymorphs, prodrugs, hydrates and solvates thereof, in asynergistically effective amount or synergistic combination effective totreat or reduce the duration of neutropenia and/or thrombocytopenia. Thecompositions of the invention are synergistically effective in treatingor reducing the duration of neutropenia and/or thrombocytopenia in humanand other mammalian subjects with neoplastic disease. A “synergisticallyeffective amount” as applied to compositions of the invention compriseG-CSF and a phorbol ester compound of Formula I or derivative compoundof phorbol esters of Formula I including pharmaceutically acceptablesalts, enantiomers, isomers, polymorphs, prodrugs, hydrates and solvatesthereof, is effective for shortening the duration of neutropenia and/orthrombocytopenia, which is effective in treating or reducing theduration of neutropenia and/or thrombocytopenia. The effect produced bythe combination of the present invention results in a response greaterthan G-CSF or a phorbol ester compound of Formula I or derivativecompound of phorbol esters of Formula I, alone or the sum of theirindividual effects

A synergistically effective amount of a phorbol ester of Formula I (suchas TPA) with G-CSF or a synergistically effective amount of acombination of a phorbol ester of Formula I (such as TPA) with EPO, maybe administered to a mammal in a single or multiple unit form eithersimultaneously or sequentially, in combined or separate formulation(s),with one or more secondary agents, or one or more adjunctive therapeuticagents; by an oral method (such as capsules, in liquid form, tablets,etc.), parenteral method (such as parenteral injection), or by any othermethods known in the art suitable for administering drugs to mammals.

The compositions of the invention comprise EPO and a phorbol estercompound of Formula I or derivative compound of phorbol esters ofFormula I including pharmaceutically acceptable salts, enantiomers,isomers, polymorphs, prodrugs, hydrates and solvates thereof, in asynergistically effective amount or synergistic combination effective totreat or reduce the duration of anemia. In particular, the compositionsof the invention are synergistically effective in treating or reducingthe duration of anemia in human or other mammalian subjects withneoplastic disease.

The compositions of the invention comprise an effective amount or unitdosage of a phorbol ester compound of Formula I or derivative compoundof a phorbol ester of Formula I, and G-CSF which may be formulated withone or more pharmaceutically acceptable carriers, excipients, vehicles,emulsifiers, stabilizers, preservatives, buffers, and/or other additivesthat may enhance stability, delivery, absorption, half-life, efficacy,pharmacokinetics, and/or pharmacodynamics, reduce adverse side effects,or provide other advantages for pharmaceutical use.

Effectiveness of the compositions and methods of the invention may bedemonstrated by a decrease in the duration of anemia, neutropenia and/orthrombocytopenia.

Compositions of the invention may be coordinately administered(simultaneously or sequentially, in combined or separateformulation(s)), with one or more secondary cancer treating agents, orother indicated or adjunctive therapeutic agents, including, but notlimited to, doxorubicin, vitamin D3, cytarabine, cytosine arabinoside,daunorubicin, cyclophosphamide, gemtuzumab ozogamicin, idarubicin,mercaptopurine, mitoxantrone, thioguanine, aldesleukin, asparaginase,carboplatin, etoposide phosphate, fludarabine, methotrexate, etoposide,dexamethasone, and choline magnesium trisalicylate.

Within the methods and compositions of the invention, a phorbol estercompound(s) of Formula I (such as TPA) as disclosed herein is/areeffectively formulated or administered with GCSF for treatingneutropenia, thrombcytopenia and/or related disorders. In exemplaryembodiments, TPA is demonstrated for illustrative purposes to be aneffective agent in pharmaceutical formulations and therapeutic methods,in combination with GCSF. The present disclosure further providesadditional, pharmaceutically acceptable phorbol ester compounds (such asTPA) in the form of a native or synthetic compound, including complexes,derivatives, salts, solvates, isomers, enantiomers, polymorphs, andprodrugs of the compounds disclosed herein, and combinations thereof,which are effective as therapeutic agents within the methods andcompositions of the invention.

Compositions of the invention may comprise a phorbol ester compound ofFormula I (such as TPA) encapsulated for delivery, separately ortogether with GCSF or EPO, in microcapsules, microparticles, ormicrospheres, prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively; in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules); or within macroemulsions.

As noted above, in certain embodiments the methods and compositions ofthe invention may employ pharmaceutically acceptable salts, e.g., acidaddition or base salts of the above-described phorbol ester compounds ofFormula I and/or related or derivative compounds (such as TPA). Examplesof pharmaceutically acceptable addition salts include inorganic andorganic acid addition salts. Suitable acid addition salts are formedfrom acids which form non-toxic salts, for example, hydrochloride,hydrobromide, hydroiodide, sulphate, hydrogen sulphate, nitrate,phosphate, and hydrogen phosphate salts. Additional pharmaceuticallyacceptable salts include, but are not limited to, metal salts such assodium salts, potassium salts, cesium salts and the like; alkaline earthmetals such as calcium salts, magnesium salts and the like; organicamine salts such as triethylamine salts, pyridine salts, picoline salts,ethanolamine salts, triethanolamine salts, dicyclohexylamine salts,N,N′-dibenzylethylenediamine salts and the like; organic acid salts suchas acetate, citrate, lactate, succinate, tartrate, maleate, fumarate,mandelate, acetate, dichloroacetate, trifluoroacetate, oxalate, andformate salts; sulfonates such as methanesulfonate, benzenesulfonate,and p-toluenesulfonate salts; and amino acid salts such as arginate,asparginate, glutamate, tartrate, and gluconate salts. Suitable basesalts are formed from bases that form non-toxic salts, for examplealuminum, calcium, lithium, magnesium, potassium, sodium, zinc anddiethanolamine salts.

Other detailed embodiments, the methods and compositions of theinvention for employ prodrugs of phorbol esters of Formula I. Prodrugsare considered to be any covalently bonded carriers which release theactive parent drug in vivo. Examples of prodrugs useful within theinvention include esters or amides with hydroxyalkyl or aminoalkyl as asubstituent, and these may be prepared by reacting such compounds asdescribed above with anhydrides such as succinic anhydride.

For instance, in current AML therapeutic regimen, G-CSF has been acommon adjuvant drug for reducing the duration of neutropenia, but notthrombocytopenia, after chemotherapy. The present invention indicatesthat G-CSF combined with phorbol esters such as TPA can treat or reducethe duration of both neutropenia and/or thrombocytopenia through thefollowing two mechanisms.

1) TPA stimulates the upstream stem cells to differentiate intodownstream stem cells. TPA also stimulates downstream stem cells. GCSFonly stimulates the downstream stem cells.

2) TPA stimulates the growth of stromal cells, which nourish the stemcells.

For example, the duration of neutropenia after high dose chemotherapyand treatment with GCSF is, for example, about 24±3 days. Thecombination of TPA and GCSF reduces the duration of neutropenia to about15±3 days, or about a 25% to 50% reduction in the duration ofneutropenia.

The combination of TPA and GCSF may result in about a 15% to 70%reduction in the duration of cytopenia, including but not limited to,neutropenia, thrombocytopenia, and/or anemia in comparison to treatmentwith GCSF or TPA alone. More preferably, the combination results inabout at 20% to 60% reduction in the duration of cytopenia; and mostpreferably, the combination results in about a 25% to 50% reduction inthe duration of cytopenia.

Likewise, the combination of TPA and EPO may result in about a 15% to70% reduction in the duration of cytopenia, including but not limitedto, neutropenia, thrombocytopenia, and/or anemia in comparison totreatment with EPO or TPA alone. More preferably, the combinationresults in about at 20% to 60% reduction in the duration of cytopenia;and most preferably, the combination results in about a 25% to 50%reduction in the duration of cytopenia.

The invention achieves a surprisingly synergistic effect by stimulatingupstream stem cells to differentiate into downstream stem cells, asshown in FIG. 1.

Alternatively, effectiveness of the compositions and methods of theinvention may also be demonstrated, for example, by an increase towardnormal levels of red blood cells, white blood cells, neutrophils, and/orplatelets. For instance, effectiveness of the compositions and methodsof the invention may be demonstrated by a decrease in neutropenia,anemia, and/or thrombocytopenia.

Effectiveness may be demonstrated using, for example, a complete bloodcount (CBC). The measurements taken in a CBC include a white blood cellcount (WBC), a red blood cell count (RBC), the red cell distributionwidth, the hematocrit, and the amount of hemoglobin. An effective amountof a composition of the present invention will increase the levelsmeasured in a complete blood count by 10%, 20%, 30%, 50% or greaterincrease, up to a 75-90%, or 95% or greater. Effective amounts will alsomove the blood protein of an individual towards the optimal category foreach type of protein.

A normal erythrocyte (RBC) count is from 4.0×10¹²/l to 5.2××10¹²/l (infemales) and from 4.4×10¹²/l to 5.7×10¹²/l (in males). Effectiveness ofthe compositions and methods herein will increase the RBC count towardsthe normal count range.

A normal hemoglobin level is typically from 130 g/l to 175 g/l.Specifically, the normal hemoglobin level is typically from 140 g/l to180 g/l in human males, and the normal hemoglobin level is typicallyfrom 120 g/l to 160 g/l in human females. Anemia is a decrease in theamount of RBCs or hemoglobin in the blood. Anemia in men is based on ahemoglobin of less than 130 to 140 g/L (13 to 14 g/dL), while anemia inwomen is less than 120 to 130 g/L (12 to 13 g/dL). Effectiveness of thecompositions and methods herein will increase the hemoglobin leveltowards the normal hemoglobin level.

A normal hematocrit level is from 0.370 to 0.460 (in females) and isfrom 0.420 to 0.520 (in males). Effectiveness of the compositions andmethods herein will increase the hematocrit level towards the normalrange.

A normal WBC count is from 4.0×10⁹/l to 10.0××10⁹/l. Effectiveness ofthe compositions and methods herein will increase the WBC count towardsthe normal count range.

Effectiveness of the compositions and methods herein may be evaluatedusing, an absolute neutrophil count (ANC). A normal ANC is between 1,500to 8,000/mm³. Individuals suffering from neutropenia have an ANC below1500/mm³, and may even reach levels below 500/mm³. Effective amounts ofthe compositions and methods herein will increase an individual's ANC by10%, 20%, 30%, 50% or greater increase, up to a 75-90%, or 95% orgreater. Effective amounts may increase ANC levels above 1500/mm³.

Effectiveness of the compositions and methods herein may further beevaluated using, for example, a platelet count. A platelet count isnormally between 150,000 to 450,000 platelets per microliter(×10-6/Liter). Individuals suffering from thrombocytopenia may haveplatelet counts below 100,000 per microliter (100,000/μl). Effectiveamounts of the compositions and methods herein will increase anindividual's platelet count by 10%, 20%, 30%, 50% or greater increase,up to a 75-90%, or 95% or greater. Effective amounts may increaseplatelet levels above 100,000 per microliter.

Effectiveness of the compositions and methods herein may additionally beevaluated, for example, by measuring the number of myeloblasts.Myeloblasts normally represent less than 5% of the cells in the bonemarrow but should not be present in circulating blood. Effective amountsof the compositions and methods herein will decrease the number ofmyeloblasts by 10%, 20%, 30%, 50% or more, up to a 75-90%, 96% orgreater decrease. Effective amounts may decrease myeloblasts to below5%.

Effectiveness of the compositions and methods herein may further beevaluated by examining myeloblasts for the presence of Auer rods.Effective amounts of the compositions of the present invention willdecrease the number of Auer rods visible by 10%, 20%, 30%, 50% or more,up to a 75-90%, 96% or greater decrease up to the complete eliminationof Auer rods.

Effectiveness of the compositions and methods of the invention may bedemonstrated by a decrease in the symptoms that accompany cytopenia,including but not limited to, neutropenia, anemia, and/orthrombocytopenia.

Effective amounts of a phorbol ester compound or related or derivativecompound of Formula I (e.g., a unit dose comprising an effectiveconcentration/amount of TPA, or of a selected pharmaceuticallyacceptable salt, isomer, enantiomer, solvate, polymorph and/or prodrugof TPA) will be readily determined by those of ordinary skill in theart, depending on clinical and patient-specific factors. Suitableeffective unit dosage amounts of the active compounds for administrationto mammalian subjects, including humans, may range from about 10 toabout 1500 μg, about 20 to about 1000 μg, about 25 to about 750 μg,about 50 to about 500 μg, about 150 to about 500 μg, about 125 μg toabout 500 μg, about 180 to about 500 μg, about 190 to about 500 μg,about 220 to about 500 μg, about 240 to about 500 μg, about 260 to about500 μg, about 290 to about 500 μg. In certain embodiments, the diseasetreating effective dosage of a phorbol ester compound or related orderivative compound of Formula I may be selected within narrower rangesof, for example, 10 to 25 μg, 30-50 μg, 75 to 100 μg, 100 to 300 μg, or150 to 500 μg. These and other effective unit dosage amounts may beadministered in a single dose, or in the form of multiple daily, weeklyor monthly doses, for example in a dosing regimen comprising from 1 to5, or 2 to 3, doses administered per day, per week, or per month. In oneexemplary embodiment, dosages of 10 to 30 μg, 30 to 50 μg, 50 to 100 μg,100 to 300 μg, or 300 to 500 μg, are administered one, two, three, four,or five times per day. In more detailed embodiments, dosages of 50-100μg, 100-300 μg, 300-400 μg, or 400-600 μg are administered once or twicedaily. In a further embodiment, dosages of 50-100 μg, 100-300 μg,300-400 μg, or 400-600 μg are administered every other day. In alternateembodiments, dosages are calculated based on body weight, and may beadministered, for example, in amounts from about 0.5 μg/m² to about 300μg/m² per day, about 1 μg/m² to about 200 μg/m², about 1 μg/m² to about187.5 μg/m² per day, about 1 μg/m² per day to about 175 μg/m² per day,about 1 μg/m² per day to about 157 μg/m² per day about 1 μg/m² to about125 μg/m² per day, about 1 μg/m² to about 75 μg/m² per day, 1 μg/m² toabout 50/μg/m² per day, 2 μg/m² to about 50 μg/m² per day, 2 μg/m² toabout 30 μg/m² per day or 3 μg/m² to about 30 μg/m² per day.

In other embodiments, dosages may be administered less frequently, forexample, 0.5 μg/m² to about 300 μg/m² every other day, about 1 μg/m² toabout 200 μg/m², about 1 μg/m² to about 187.5 μg/m² every other day,about 1 μg/m² to about 175 μg/m² every other day, about 1 μg/m² per dayto about 157 μg/m² every other day about 1 μg/m² to about 125 μg/m²every other day, about 1 μg/m² to about 75 μg/m² every other day, 1μg/m² to about 50 μg/m² every other day, 2 μg/m² to about 50 μg/m² everyother day, 2 μg/m² to about 30 μg/m² per day or 3 μg/m² to about 30μg/m² per day. In additional embodiments, dosages may be administered 3times/week, 4 times/week, 5 times/week, only on weekdays, only inconcert with other treatment regimens, on consecutive days, or in anyappropriate dosage regimen depending on clinical and patient-specificfactors.

Erythropoietin is a glycosylated protein hormone and a haematopoieticgrowth factor produced primarily in the kidneys, and for clinical use,is produced by recombinant DNA technology and the name epoetin is oftenapplied to such material. Seenoblood.org/forum/content/179-erythropoietin_-28epo-29. Epoetin alfa,epoetin beta, epoetin gamma, epoetin omega, and epoetin zeta arerecombinant human erythropoietins derived from a cloned humanerythropoietin gene; all of which have the same 165 amino acid sequencebut differ in the glycosylation pattern. Id. Epoetin delta is arecombinant human erythropoietin derived from a genetically engineeredcontinuous human cell line, and has the same amino acid sequence andglycosylation pattern as human erythropoietin. Id.

EPO such as EPOETIN® may be given either as an IV or SC injection, asdescribed atinceptapharma[dot]com/epoetin/submenu_page_view.php?menu_id=86&submenu_id=223&fs,the disclosure of which is herein incorporated by reference in itsentirety. For instance, the dosage may be adjusted for each patient toachieve and maintain hemoglobin levels between 10 to 12 g/dL. Id. Forexample, if hemoglobin is increasing and approaching 12 g/dL, the dosemay be reduced by approximately 25%; if the hemoglobin continues toincrease, the dose may be temporarily withheld until the hemoglobinbegins to decrease and then reinitiated at a dose approximately 25%below the previous dose; or if the hemoglobin increases by more than 1g/dL in a 2-week period, the dose may be decreased by approximately 25%.Id. If the increase in the hemoglobin is less than 1 g/dL over 4 weeksand iron stores are adequate, the dose of EPOETIN® may be increased byapproximately 25% of the previous dose. Further increases may be made at4-week intervals until the specified hemoglobin is obtained. Id.

The dose of EPO may be titrated for each patient on chemotherapy or whohave undergone chemotherapy, to achieve and maintain the lowesthemoglobin level sufficient to avoid the need for blood transfusion andnot to exceed the upper safety limit of 12 g/dL.inceptapharma[dot]com/epoetin/submenu_page_view.php?menu_id=86&submenu_id=223&fsThe initial recommended dose of EPO in adults is 150 Units/kg SC TIW or40,000 Units SC Weekly, and the initial recommended dose of EPO inpediatric patients is 600 Units/kg IV weekly. Id.

Suitable effective unit dosage amounts of erythropoeitin may depend onseveral factors and will be within the discretion of the subject'sphysician. For example, some patients may be more or less sensitive tothe compounds or compositions described herein, and for those patientscompositions providing a higher of a lower plasma or serum value may bepreferred. Also, some subjects may metabolize the compound or maymetabolize it at different rates, and so dosages and/or alternativedosage forms may be required to provide the desired serum or plasmaconcentration. Skilled artisans will appreciate that specific dosages ofEPO in compositions of the present invention may be adjusted dependingon conditions of disease, the age, body weight, general healthconditions, sex, and diet of the subject, dose intervals, administrationroutes, excretion rate, and combinations of active compounds.

The dosing regimen for EPO may include doses such as 75 to 150 IU forevery kilogram (u/kg) of body weight given daily or every other day; 600u/kg given once a week; or 300 u/kg three or four times a week; asdescribed at noblood.org/forum/content/179-erythropoietin_-28epo-29 is asuggested dosing guide, the disclosure of which is herein incorporatedby reference in its entirety. For instance, for a 70 kg patient, 60,000IU per week may be ordered. Id.

Suitable effective unit dosage amounts of EPO may include a range from450 IU/kg to 900 IU/kg, given daily or every other day, or given once,twice, three times or four times a week. See,noblood.org/forum/content/179-erythropoietin_-28epo-29

Suitable effective unit dosage amounts of EPO-beta may include 1000IU/0.3 mL, 2000 IU/0.3 mL, 3000 IU/0.3 mL, 4000 IU/0.3 mL, 5000 IU/0.3mL, 6000 IU/0.3 mL, 10,000 IU/0.6 mL, and 30,000 IU/0.6 mL solutions;and contains urea, sodium chloride, sodium phosphate, and water, inpre-filled syringes for injection. Seenoblood.org/forum/content/179-erythropoietin_-28epo-29.

Epoetin alfa may be administered by injection of 1 mL of a water-basedsolution which may contain a single dose of 2000, 3000, 4000, 10,000, or40,000 units of epoetin alfa per single dose, along with otheringredients including albumin, based on treatment requirements andweight of patient. Seenoblood.org/forum/content/179-erythropoietin_-28epo-29. In addition,multidose injections may also be administered with 10,000 units or20,000 units per 1 mL of injection solution. Id. This applies to otherforms of EPO. Id. Chronic diseases, such as renal failure, heartdisease, diabetes, and inflammatory diseases like rheumatoid arthritis,all contribute to anemia and produce a blunted response to EPOtherapy—in all such cases the dosage should be increased. Id.

Dosages of EPO alfa (rch) or EPREX® may be administered as described atmedsafe.govt.nz/profs/datasheet/e/eprexinj.pdf, the disclosure of whichis herein incorporated by reference in its entirety. For example, EPOalfa may be administered subcutaneously with 150 units/kg 3 timesweekly, or 40,000 units weekly, and/or 300 units/kg 3 times weekly or60,000 units weekly. Seedrugs.com/ppa/epoetin-alfa-erythropoietin-epo.html, the disclosure ofwhich is herein incorporated by reference in its entirety.

The dosage of EPO may include high doses as described in U.S. Pat. No.7,232,797, the disclosure of which is herein incorporated by reference.For instance, U.S. Pat. No. 7,232,797 describes a dosage of EPO of 5000IU/kg weekly or 17,000˜25,000 IU/kg (biweekly or triweekly).

The dosage of EPO may include low doses as described in CA2418531, thedisclosure of which is herein incorporated by reference. For instance,the CA2418531 patent describes dosage of EPO from about 1 to about 90IU/kg per week; as well as an initial treatment dose of about 75 toabout 120 IU/Kg per week and maintenance dose of about 20 to about 75IU/Kg per week. In addition, CA2418531 describes administration ofrecombinant Epoetin Omega at a dose of 5-150 IU/Kg, one to three timesper week.

An effective dose or multi-dose treatment regimen for the instantdisease treating (alternatively, “neutrophil stimulating,”“erythropoiesis stimulating,” or “platelet stimulating”) formulations ofthe invention will ordinarily be selected to approximate a minimaldosing regimen that is necessary and sufficient to substantially treator reduce/shorten the duration of anemia, neutropenia, and/orthrombocytopenia in the subject. A dosage and administration protocolwill often include repeated dosing therapy over a course of several daysor even one or more weeks or years. An effective treatment regime mayalso involve prophylactic dosage administered on a day or multi-dose perday basis lasting over the course of days, weeks, months or even years.

Effectiveness of the compositions and methods of the invention may alsobe demonstrated by a decrease in the symptoms of subjects suffering fromneoplastic disease including, but not limited to, anemia, chronicfatigue; excessive or easy bleeding, such as bleeding of the nose, gums,and under the skin; easy bruising, particularly bruising with noapparent cause; shortness of breath; petechiae; recurrent fever; swollengums; slow healing of cuts; bone and joint discomfort; recurrentinfections; weight loss; itching; night sweats; lymph node swelling;fever; abdominal pain and discomfort; disturbances in vision; coughing;loss of appetite; pain in the chest; difficulty swallowing; swelling ofthe face, neck and upper extremities; a need to urinate frequently,especially at night; difficulty starting urination or holding backurine; weak or interrupted flow of urine; painful or burning urination;difficulty in having an erection; painful ejaculation; blood in urine orsemen; frequent pain or stiffness in the lower back, hips, or upperthighs; and/or weakness.

Effectiveness of the compositions and methods of the invention in thetreatment of rheumatoid arthritis may also be demonstrated by a changein the erythrocyte sedimentation rate. An effective amount of thecompositions of the invention would decrease the levels of erythrocytesedimentation by 10%, 20%, 30%, 50% or more, up to a 75-90%, 96% orgreater decrease over the initial diagnostic levels of erythrocytesedimentation. Effectiveness may also be demonstrated by a change in thelevels of rheumatoid factor and anti-cyclic citrullinated antibodies.

The compounds and compositions described herein can be formulated intopharmaceutically acceptable compositions, which may include one or morepharmaceutically acceptable carriers. Such compositions may be preparedby mixing one or more compounds or compositions described herein,including, e.g., pharmaceutically acceptable salts thereof orstereoisomers thereof, with pharmaceutically acceptable carriers,excipients, binders, diluents or the like to treat or reduce theduration of cytopenia such as neutropenia, thrombocytopenia, and/oranemia.

The instant compositions can be formulated for various routes ofadministration, for example, by oral, transdermal, parenteral, rectal,nasal, vaginal administration, or via implanted reservoir or otherdevice such as a stent. Such implants may employ known inert materialssuch as silicones and biodegradable polymers. They also may be providedin combination with delivery vehicles such as in micelles or liposomes,or some other encapsulating technology. Parenteral or systemicadministration includes, but is not limited to, subcutaneous,intravenous, intraperitoneally, intramuscular, intrathecal,intracranial, and intracerebroventricular injections.

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, and caplets are acceptableas solid dosage forms. These can be prepared, for example, by mixing oneor more compounds disclosed herein, or pharmaceutically acceptable saltsor stereoisomers thereof, with at least one additive, including but notlimited to, sucrose, lactose, cellulose sugar, mannitol, maltitol,dextran, starch, agar, alginates, chitins, chitosans, pectins,tragacanth gum, gum arabic, gelatins, collagens, casein, albumin,synthetic or semi-synthetic polymers and glycerides. Optionally, oraldosage forms can contain other ingredients to aid in administration,such as an inactive diluent, lubricants such as magnesium stearate,preservatives such as paraben or sorbic acid, anti-oxidants such asascorbic acid, tocopherol or cysteine, a disintegrating agent, binders,thickeners, buffers, sweeteners, flavoring agents or perfuming agents.Tablets and pills may be further coated with coating materials known inthe art.

Liquid dosage forms for oral administration may be in the form ofpharmaceutically acceptable emulsions, syrups, elixirs, suspensions, andsolutions, which may contain an inactive diluent, such as water.Pharmaceutical formulations and medicaments may be prepared as liquidsuspensions or solutions using a sterile liquid, including, but notlimited to, oil, water, alcohol, and combinations thereof.Pharmaceutically suitable surfactants, suspending agents, emulsifyingagents, may be added for oral or parenteral administration.

Injectable dosage forms include aqueous suspensions or oil suspensionswhich may be prepared using a suitable dispersant or wetting agent and asuspending agent. Injectable forms may be in solution phase or in theform of a suspension, which is prepared with a solvent or diluent,including but is not limited to, sterilized water, Ringer's solution, oran isotonic aqueous saline solution. For injection, the pharmaceuticalformulation and/or medicament may be a powder suitable forreconstitution with an appropriate solution, and may optionally containstabilizers, pH modifiers, surfactants, bioavailability modifiers andcombinations thereof. Examples of such suitable powders include, but arenot limited to, freeze dried, rotary dried or spray dried powders,amorphous powders, granules, precipitates, or particulates.

Compounds and compositions described herein also may be administered tothe lungs by inhalation through the nose or mouth. Suitablepharmaceutical formulations for inhalation include but are not limitedto, aqueous and nonaqueous aerosols, solutions, sprays, dry powders, oraerosols containing any appropriate solvents and optionally othercompounds such as, but not limited to, stabilizers, antimicrobialagents, antioxidants, pH modifiers, surfactants, bioavailabilitymodifiers and combinations thereof. Formulations for inhalationadministration may contain excipients including but not limited to,lactose, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate.An aqueous aerosol is made by formulating an aqueous solution orsuspension of the compound or composition together with conventionalpharmaceutically acceptable carriers and stabilizers, which include butare not limited to, nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols may be prepared from isotonicsolutions, or a nonaqueous suspension (e.g., in a fluorocarbonpropellant) can also be used to deliver embodiments of the compounds andcompositions described herein.

The compounds and compositions of the present invention may be providedin a spray, nasal drops or aerosol containing an appropriate solvent(s)and optionally other compounds such as, but not limited to, stabilizers,antimicrobial agents, antioxidants, pH modifiers, surfactants,bioavailability modifiers and combinations thereof, for nasaladministration.

Compounds and compositions of the present invention may be provided forsustained or immediate release. Sustained release dosage forms controlthe rate of release, and can maintain an effective concentration of thecomposition over time, thereby providing the recipient with atherapeutic effect over an extended duration. The pharmaceuticalcomposition is a dosage form selected from the group consisting of atablet, liquid for oral administration, oral spray, intranasal spray,inhalable formulation, pill, gel, solid, capsule, multi-particulate,transdermal patch, implantable dosage, and injectable solution includingintravenous drip (including in lyophilized and re-constituted form); aswell as dosage forms that swell or unfold so that the dosage form isretained in the stomach or the upper portion of the small intestine forat period of least 1 hour, at least 2 hours, at least three hours, atleast 4 hours, at least 5 hours, at least 6 hours or for a period oflonger than 6 hours. Examples of patents that describe sustained releasecompositions include, but are not limited to, U.S. Pat. Nos. 7,438,927,7,413,751, 7,405,238, 6,723,340, 6,682,759, 6,635,280, 6,488,962,6,451,808, 6,340,475, 5,972,389, 5,582,837, and 5,007,790.

The following non-limiting examples are provided merely to illustratevarious aspects or embodiments of the present invention.

EXAMPLES Example 1 In Vitro Study of TPA and GCSF on Colony FormingCells

The combination of TPA and GCSF generated stronger stimulating effectson colony forming cells than TPA or G-CSF alone, as shown in FIG. 2.

Myelosuppression is the most common adverse reactions of cancer patientswho use chemotherapy drugs, severe bone marrow suppression often makeschemotherapy difficult to continue as planned, may be bring out thecomplications, could be life-threatening. Recently, rhG-CSF, EPO areused widely to treat leukopenia or anemia which coursed by chemotherapyand radiotherapy. But only rhG-CSF, EPO can not recover medullaryhematopoiesis in the short term for those who accepted high intensity ormany times chemotherapy, especially for leukemia patients. Becausechemotherapy drugs can hurt the normal hematopoietic cells andmicroenvironment in the bone marrow, when they are killing cancer cells.Meanwhile rhG-CSF, EPO play a role in the downstream of hematopoiesis.Such as G-CSF effects on myeloid progenitor stage, which stimulate theirproliferation, differentiation and promote mature neutrophils to bereleased into the peripheral blood. EPO play a role on erythroidprogenitor cells stage, to stimulate erythropoiesis, increase the numberof red blood cells in peripheral blood. But they have no effects on bonemarrow microenvironment all. Therefore, it is important to find a way toquickly restore bone marrow hematopoiesis. It is reported that12-O-tetradecanoylphorbol-13-acetate which is called phorbol ester(TPA), not only can induce a variety of leukemia cells to normal cells,but also TPA have a certain influence on bone marrow hematopoietic andcan increase white blood cell.

To explore the effect of TPA alone or combined rhG-CSF on bone marrowhematopoietic cells proliferation and colony formation ability whichfrom patients of acute myeloid leukemia (AML) in the period of bonemarrow suppression in vitro and to observe the effect of TPA on bonemarrow stromal cells (BMSCs) proliferation or inhibition from patientsof AML in the period of myelosuppression and the healthy persons.

Methods:

-   1) Human bone marrow cells from the same AML patient after    chemotherapy were cultured by methyl cellulose semi-solid culture    medium. Groups: Blank, TPA (10 ng/ml), G-CSF (50 ng/ml), TPA (10    ng/ml)+GCSF (50 ng/ml). The experiments were repeated for 4 times.-   2) The stroma cells of bone marrow from the healthy human and the    AML patient after chemotherapy were cultured by methyl cellulose    semi-solid culture medium. Different TPA concentrations were added.    Groups: Blank, TPA (0.1 ng/ml), TPA (1.0 ng/ml), TPA (5 ng/ml), TPA    (10 ng/ml), TPA (20 ng/ml), TPA (30 ng/ml).-   3) Cultivation of BMSCs from healthy persons and patients of AML in    the bone marrow suppression phase in vitro, added to TPA of    different concentrations, 0.1 ng/ml, 1.0 ng/ml, 5 ng/ml, 10 ng/ml,    20 ng/ml, 30 ng/ml, and set up a control to detect cell    proliferation or inhibition with CCK8 method.

Results and Conclusion:

1. Compared the clones of four groups in incomplete medium, in thecontrol group a small cell clusters can be seen for 24-72 hours, but thecells were dead as time prolong. Cultivated 14 days, control group haveno clones formation, CFU-GM are dominated for G-CSF group, TPA alone andcombination with G-CSF group have myeloid colony formation, while BFU-Eand CFU-GEMM. For G-CSF group, TPA alone group and combination withG-CSF group, the total number of clones were higher than control group(both P<0.05), meanwhile the clones for TPA alone group and combinationwith G-CSF group are higher than G-CSF group (both P<0.05), the clonesof joint group is higher than that of TPA group (P<0.001).

2. Compared the clones of different concentration of TPA stimulation inincomplete medium, the total clones of 5 ng/ml, 10 ng/ml, 20 ng/ml groupare higher than other groups, including 10 ng/ml in the highest, thereare statistical significance (both P<0.05). While decreasing orincreasing TPA concentration can not improve the colony number. Thebetter concentration of TPA to stimulate the colony formation is 5˜20ng/ml. Cultivated 14 days, except CFU-GM, CFU-GEMM were seen in 1 ng/ml,5 ng/ml, 10 ng/ml, 20 ng/ml group, and can be seen fewer BFU-E in 5ng/ml, 10 ng/ml, 20 ng/ml group, while the other group concentration didnot see the erythroid clones.

3. Compared the clones of different concentration of TPA and G-CSF (50ng/ml) stimulation in incomplete medium. Combination with G-CSF thebetter concentration of TPA is 1˜10 ng/ml (both P<0.05). Combinationwith G-CSF the best concentration of TPA is 5 ng/ml and 1 ng/ml forCFU-GM and CFU-GEMM. The main clone types are myeloid colones, CFU-GM,also have CFU-GEMM and fewer BFU-E.

4. Compared the clones of four groups in complete medium, clones can beseen in four groups including of CFU-GM, CFU-GEMM, BFU-E, CFU-E. Theclones of TPA with G-CSF group are highest compared with TPA group,G-CSF group and control group, difference have statistical significance(both P<0.05). While TPA group and G-CSF group the number of clones ofTPA group and G-CSF group have no statistical significance (P=0.577).

5. Experiments show that TPA promote bone marrow stromal cells fromhealthy persons proliferation of concentration 5˜10 ng/ml (cell number2×105/ml). Decreasing or increasing the concentration of TPA, it showsthat BMSCs are inhibited. Meanwhile, TPA promote bone marrow stromalcells from the AML patients proliferation of concentration 5˜30 ng/ml(cell number 2×105/ml). 5 ng/ml of TPA is the best concentration ofpromoting proliferation, and G-CSF had no effect on the growth of BMSCsfrom healthy persons or AML patients.

Conclusion:

TPA promoted hematopoietic cell clone formation lonely, mainly of CFU-GMfor bone marrow of AML patients in the period of bone marrow suppressionin vitro, the best concentration is 10 ng/ml. TPA promoted the formationof CFU-GM, CFU-GEMM, and BFU-E at different stages.

TPA and G-CSF have synergistic effects in promoting bone marrow myeloidclones formation, in addition promote CFU-GEMM and BFU-E formation, thebest concentration of TPA is 5 ng/ml. G-CSF promoted the formation ofCFU-GM, but had no effect on CFU-GEMM and BFU-E.

TPA promoted the growth of the stroma cells of bone marrow (BMSCs) ofthe healthy human and the AML patient after chemotherapy, in the bonemarrow suppression phase in vitro. The optimal concentrations were 5ng/ml and 10 ng/ml.

As shown in FIG. 1, TPA stimulated the upstream stem cells todifferentiate into downstream stem cells. GCSF only stimulated thedownstream stem cells. TPA also stimulates downstream stem cells.Neutrophils are one type of granulocytes.

Example 2 Project TPA (PD616) for AML

MOA: Protein kinase C (PKC) activator

Indication:

-   -   1) current protocol: salvage therapy of AML after relapse    -   2) new strategy: AML supportive care after        induction/consolidation chemotherapy on WBC and platelet        recovery.

Rationale:

-   -   (1) Activation of PKC facilitates hematopoietic cells recovery    -   (2) PKC induces differentiation of leukemia cells

TPA in AML Strength

-   -   To enhance bone marrow recovery in 1 L AML after induction and        consolidation chemotherapy    -   Well established mechanism of action by activation of PKC    -   Well known tolerable toxicity profile from prior clinical        studies    -   Strong efficacy data in AML patients with potential shortening        of neutropenia & thrombocytopenia duration from 20 to 12 days        -   Decrease hospital stay        -   Minimize chance of infection        -   Decrease the need of blood product support        -   Reduce AML patient care cost

TPA as a supportive care for white cell & platelets recovery after AMLchemotherapy

-   -   Pitfalls of standard of care (SOC): G-CSF or GM-CSF does not        work on early hematopoietic progenitor cells and with limited        efficacy in shortening of the duration of neutropenia &        thrombocytopenia after induction or consolidation therapy.    -   There is no effective approach to facilitate platelet recovery        after chemotherapy in AML induction/consolidation.    -   TPA enhances growth of early progenitor cells and potentially        helps shorten the duration of neutropenia and thrombocytopenia    -   Annual incidence 14,000 cases of AML, and 80% will receive        aggressive chemo and develop prolonged neutropenia.    -   Target:        -   shortening of duration of neutropenia and hospital stay from            20 to 12 days,        -   Decrease blood products (PRBC and platelets) support, due to            facilitation of platelet recovery        -   Decrease infection complication        -   No negative impact on efficacy, CR rate or duration of            response

Indication

-   -   Shortening of neutropenia & thrombocytopenia in AML    -   patients after induction or consolidation chemotherapy.        Shortening of neutropenia and thrombocytopenia.

Administration

-   -   TPA IV 3 times per week (M-W-F) until ANC over 1000 and        platelets over 20,000 for at least 2 days at 0.125 mg/m² as a        single course starting 24-48 hours after completion of        chemotherapy whereas other supportive care remains as SOC.

Efficacy

-   -   Decrease the duration of neutropenia by 40%, from 20 days to 12        days. Decrease average hospitalization days by 40%. Decrease        blood product support by 40%.

Safety

-   -   All toxicities not significantly worse than common toxicities        associated with standard AML chemotherapy.

Competition

-   -   G-CSF and GM-CSF but not very effective    -   Phase 2 single arm study of 12 patients, TPA starts at 24-48        hours after completion of standard chemotherapy (6 for induction        3+7 and 6 for consolidation with 2+5 or high-dose Ara-C). TPA IV        for M-W-F per week until ANC over 1000 and platelet over 20,000        persistently for 2+ days, whereas other supportive care same as        SOC.    -   Double-blinded randomized phase 2, TPA+G-CSF vs. G-CSF in 1:1.        20 patients sample size with ˜90% power and alpha 0.1, to detect        a decrease of the duration of neutropenia & thrombocytopenia by        40% (from 20 days to 12 days)        -   Primary endpoints: Duration of neutropenia, blood product            supports and hospitalization date all decreased by 40%.    -   Target goal of 40% reduction is achieved, and no obvious        unfavorable effect to leukemia therapy.        Additional AML protocol includes starting TPA at 24-48 hours        after completion of chemotherapy and watch duration of        neutropenia & thrombocytopenia        Randomized phase 2 trial in two cohorts. One for induction        chemotherapy, one for consolidation chemotherapy. Show duration        of neutropenia decreased by 40%.

Example 3

This protocol is induction therapy, and does not include consolidation.This adjuvant therapy combines TPA and G-CSF (Granulocyte colonystimulating factor).

This is a single-arm, open label study. Ten (10) patients receivestandard induction chemotherapy with idarubicin (12 mg/m2) ordaunorubicin (60 mg/m2) on days 1, 2, 3 and Ara-C continuous (100-200mg/m2/day) infusion on days 1-7. On Day 8 or 24 hours after completionof all scheduled Ara-C infusion, TPA is started at 0.125 mg/m² IV everytwo days, in addition to G-CSF (400 μg subcutaneously daily) supportuntil absolute neutrophil count (ANC) above 1000/μL for two consecutivedays.”

A Phase 2a study of phorbol ester in shortening the duration ofneutropenia and thrombocytopenia in acute myelocytic leukemia patientswho receive induction chemotherapy.

Phorbol ester (12-O-Tetradecanoylphorbol-13-acetate, TPA) is an agonistof protein kinase C, and has been shown to increase early hematopoieticprogenitor cells by in vitro studies. In prior Phase 1 dose escalationstudies of TPA, TPA is observed to be capable of shortening the durationof post-chemotherapy neutropenia in acute myelocytic leukemia (AML)patients. In the recommended Phase 2 dose at 0.125 mg/m² daily up to 5days a week×2 weeks, it is well tolerated with only minor adverse eventssuch as shortness of breath, proteinuria, fever, chills and irritationof vein at infusion site. This study is designed to examine the efficacyof TPA as a supportive care agent to enhance bone marrow recovery in AMLpatients after induction chemotherapy.

Study objectives:

-   -   1. Evaluate the safety and tolerability of TPA in AML patients        after induction chemotherapy    -   2. Evaluate preliminary efficacy in shortening of the duration        of neutropenia and thrombocytopenia in AML patients after        induction chemotherapy    -   3. Evaluate the preliminary Complete Remission (CR) rate after        induction chemotherapy with maintenance TPA after induction        therapy.

Eligibility

The inclusion criteria include:

-   -   1. Patients diagnosed with AML or advanced myelodysplastic        syndrome (MDS, such as refractory anemia with excess blasts        (RAEB), or RAEB with transformation), if their bone marrow blast        count is over 20%.    -   2. AML should be classified by FAB classification, and all        subtypes are allowed and recorded during enrollment, except        patients with M3 or acute promyelocytic leukemia.    -   3. AML or advanced MSD patients who are considered suitable to        receive 3+7 induction chemotherapy (anthracycline and        cytarabine)    -   4. Age 18-70    -   5. ECOG 0-2    -   6. No evidence of major organ dysfunctions as defined by        Creatinine ≤2 mg/dL, AST/ALT≤5×ULN, Bilirubin≤2 mg/dL, and no        major cardiovascular problems such as recent acute myocardiac        infarction or stroke within 6 months from enrollment.    -   7. Patients with adequate cardiac function without history of        congestive heart failure as defined by no worse than American        Heart Association class I (Patients with cardiac disease but        resulting in no limitation of physical activity. Ordinary        physical activity does not cause undue fatigue, palpitation,        dyspnea or anginal pain).    -   8. Patients able to give consent for the study

The exclusion criteria include:

-   -   1. Patients with other non-AML malignancies within the past 24        months, except those that are considered curable, such as        treated basal cell carcinoma of skin, resected early stage        malignancies such as ductal carcinoma in situ of breast and        other cured cancers.    -   2. Patients with clinical active or chronic infection and not        suitable for standard AML 3+7 induction therapy    -   3. Patients with recent major bleeding, surgery and other major        medical problems within 6 months who are not suitable for        standard AML 3+7 induction therapy.    -   4. Patients with chronic COPD who require chronic oxygen        supplement to maintain pulse oxygen saturation above 92%.    -   5. Lactating and pregnant women    -   6. Patients with known positive HIV infection in the past

Study Design

This is a single-arm, open label study. Ten (10) patients receivestandard induction chemotherapy with idarubicin (12 mg/m²) ordaunorubicin (60 mg/m²) on days 1, 2, 3 and Ara-C continuous (100-200mg/m²/day) infusion on days 1-7. On Day 9 or 24 hours after completionof all scheduled Ara-C infusion, TPA is started at 0.125 mg/m² IV everymorning for 5 days on then 2 days off. Same 5 days on and 2 days offcycle of TPA administration is repeated once until patients' absoluteneutrophil count (ANC) is above 1000/μL for two consecutive days. G-CSFat 400 μg subcutaneously or intravenously daily is started at the sameday as the first day TPA starts but is administered in the afternoon, orapproximately 8 hours after the morning dose of TPA. G-CSF is alsostopped when patients' absolute neutrophil count (ANC) is above 1000/μLfor two consecutive days. This sequential approach of administration ofTPA followed by G-CSF is designed based on TPA stimulation of theproliferation of early progenitors such as CFU-GM and CFU-GEMM; whereasG-CSF stimulates the proliferation of later progenitor mainly CFU-GM orCFU-G. Without the expansion of early progenitor population, G-CSF wouldnot have the target cell population and work effectively to enhance therecovery of normal white blood cells.

All other supportive care such as IV broad-spectrum antibiotics,anti-viral (such as anti-herpetic agents), and anti-fungal (such asanti-Candidiasis agents) support follow the standard practice guidelinefor AML induction therapy. Blood product support also follows thestandard practice guideline with transfusion of packed red cells (PRBC)when hematocrit is below 30 and platelet count below 10,000 if noclinical evidence of bleeding (or 50,000 if clinical evidence ofbleeding). All patients are hospitalized for the whole induction perioduntil ANC and platelet recovery to adequate level without evidence ofactive infection. Standard care for neutropenia is adopted.

Study Duration

After patients recover from induction therapy, patients may bedischarged from the hospital and return later for subsequent additionalchemotherapy such as high dose Ara-C or considered for bone marrowtransplantation per treating physician discretion based on their riskfactors. Patients will be off study after they return for follow-up bonemarrow evaluation for the efficacy of the induction therapy. All furtherconsolidation therapy will not be considered part of the study.

Safety Evaluation

Safety analysis is evaluated based on Common Terminology Criteria forAdverse Events (CTCAE) vs. 4.0. The commonly observed adverse eventsincludes shortness of breath, fever, chills and proteinuria. Thetreatment-related fever and chills are consistent with a cytokineincrease after IV infusion of the study medication, but it is generallytransient and subsides after 24 hours. Acetaminophen is used forsymptomatic relief of the fever if necessary.

Efficacy Analysis

Evaluation of neutropenia and thrombocytopenia after inductionchemotherapy is done by daily hematological tests, including completeblood count and differential count. Day 14 bone marrow is routinely doneaccording to the standard clinical practice guideline to assess anyresidual blasts 7 days after completion of induction therapy. Flowcytometry of the aspirated bone marrow is tested to differentiate therecovering normal progenitor cells versus residual blasts. The durationof neutropenia and thrombocytopenia is assessed separately and the ANCand platelet counts should be plotted in a diagram for each patient.

Statistics Analysis

The study explores the duration of neutropenia and thrombocytopeniaafter standard 3+7 induction chemotherapy in AML patients. Usually, 80%of AML patients will have a duration of neutropenia/thrombocytopenia of20±3 days (i.e. ANC or platelet recovery at approximately at Days 24-30assuming chemotherapy starts at Day 1) and then are discharged from thehospital if there is no evidence of infection. In the study, TPAshortens the duration of neutropenia and thrombocytopenia significantly.Duration of neutropenia or thrombocytopenia for patients treated withTPA is reduced to 12 days (ANC or platelet recovery at Day 14-24 days)with a standard deviation of 5 days, the sample size of 10 patients willhave 90% power to detect a difference and reject the null hypothesis.

Example 4

Male patient, age 25, diagnosed with AML (M2). Patient's bone marrowmyeloblast plus promyelocyte count was about 60%. After he received onestandard course of DA regimen (7 Ara C+3 Daunorubicin), his peripheralWBC count dropped to 0.8×10⁹/L. He was then administered 150 μg G-CSFeach day. After chemotherapy, it took 12 days (duration of neutropeniaand thrombocytopenia) to bring both his platelet count and WBC count,including the neutrophil count, back to normal. His bone marrowmyeloblast plus promyelocyte count was about 40%, still way above thenormal value (0˜5%). Then, he received his second standard course of DAregimen. After the second course of DA regimen, his peripheral WBC countand platelet count dropped to 0.6×10⁹/L and 80×10⁹/L respectively. Hewas then administered TPA plus G-CSF (150 μg TPA followed with 150 μgG-CSF) each day of day 1 and day 2. On day 4 and day 5, he wasadministered with only 150 μg TPA each day. The WBC counts andneutrophil percentage* in parenthesis were 1.8×10⁹/L (39%) on day 3,6.5×10⁹/L (72%) on day 7, and 5.7×10⁹/L (77%) on day 14. The estimatedduration of neutropenia after chemotherapy was shortened to about 5days. The platelet counts were 330×10⁹/L on day 3, 715×10⁹/L on day 7,and 568×10⁹/L on day 14. The estimated duration of thrombocytopeniaafter chemotherapy was shortened to less than 3 days. After one morecourse of DA regimen followed with TPA plus G-CSF treatment, his bonemarrow myeloblast plus promyelocyte count was 2%, falling to the normalvalue.*Neutrophils usually make up 60 to 70% of circulating WBC.

Example 5

Male patient, age 33, was diagnosed with myelodysplastic syndromes(MDS5q−). He had been treated with therapy which included EPO, G-CSF,thalidomide, and testosterone for 7 months without any improvement. Hishematopoietic function was very low, especially erythropoiesis. Hishemoglobin level was 40 g/L without blood transfusion. Besides receivingmedication, he also had blood transfusion each month. After bloodtransfusion, his hemoglobin level reached 70-80 g/L. One to two weekslater, it dropped to 60 g/L. By the end of the month, it dropped to 40g/L again. He had to receive blood transfusion again. He had suffered aloss of working ability and could not live a normal life. He wasadministered TPA+G-CSF+EPO treatment (TPA: 150-180 μg iv infusion+G-CSF:150 μg im+EPO: 5000 unit im) 5 times. Each time, EPO and G-CSF weregiven 5 hours after TPA was given. After the 5 treatments of TPA+G-CSFwith EPO, he maintained a hemoglobin level of 70 g/L without bloodtransfusion. He has ceased blood transfusions since then. His hemoglobinlevel continued to increase gradually the following two months andreached 120 g/L in three months after TPA+G-CSF+EPO treatment, close tothe normal hemoglobin level (130-175 g/L). His WBC, RBC and plateletcounts also gradually increased to nearly normal levels. He hasrecovered his daily work and normal life.

Example 6 Drug Induced Cytopenia Treated by TPA

Ninety (90) adult mice were randomly assigned to 9 groups (10 mice pergroup). No drug was administered in Control group. Model group was givenDNR (6 mg/kg)+Ara-C (150 mg/kg) on Day 0. The rest of the 7 groups weregiven DNR (6 mg/kg)+Ara-C (150 mg/kg) on Day 0, and each group on Day 7,Day 8, and Day 9 was administered with one of the following drugs: G-CSF(10 μg/kg), EPO (500 IU/kg), TPA (12.5 μg/kg), TPA (25 μg/kg), TPA (50μg/kg), TPA (12.5 μg/kg)+GCSF (10 μg/kg), or TPA (12.5 μg/kg)+EPO (500IU/kg).

Experiment Model (3 Days after DNR+Ara-C)

TABLE 1 The comparison of blood cell counts between each group (n = 10)Dose Group (mg · kg⁻¹) WBC (×10⁹/L) RBC (×10¹²/L) PLT (×10⁹/L) Control —6.47 ± 0.39 10.05 ± 0.43 1478 ± 125 Model DNR6 + 1.03 ± 0.26**  9.91 ±0.63  342 ± 59** Ara-c150 Note: Date represent means SD. “*” P < 0.05,“**” P < 0.01 vs control group

TABLE 2 The comparison of blood cell counts between each group (n = 10)(6 days after DNR + Ara-C) Dose PLT Group (mg · kg⁻¹) WBC (×10⁹/L) RBC(×10¹²/L) (×10⁹/L) Control — 6.72 ± 0.8  9.7 ± 0.48 1432 ± 79 ModelDNR6 + Ara- 2.76 ± 0.61** 6.04 ± 0.74**  170 ± 51** c150 Note: Daterepresent means SD. “*” P < 0.05, “**” P < 0.01 vs control group

TABLE 3 The comparison of blood cell counts between each group (n = 10)(8 days after DNR + Ara-c, 1 day after 1^(st) TPA or other) Group Dose(μg · kg⁻¹) WBC (×10⁹/L) RBC (×10¹²/L) PLT (×10⁹/L) Control — 6.70 ±0.17 10.05 ± 0.24 1439 ± 14.18 Model — 3.30 ± 0.30

 5.02 ± 0.14

 98 ± 14.8

G-CSF 10  5.3 ± 0.62

 4.94 ± 0.42

 373 ± 76.27

EPO 500  3.7 ± 0.69

 7.32 ± 0.10

 385 ± 19.28

TPA 12.5  6.4 ± 0.62

 6.08 ± 0.49

 395 ± 30.41

TPA 25  6.4 ± 0.35

 7.09 ± 0.23

 391 ± 20.42

TPA 50  6.5 ± 0.75

 7.10 ± 0.24

 413 ± 9.64

TPA + GCSF 12.5 + 10   8.5 ± 0.46

 6.22 ± 0.61

 405 ± 47.62

TPA + EPO 12.5 + 500  6.8 ± 0.79

 8.61 ± 0.27

 417 ± 45.3

Note: Date represent means SD.

 P < 0.05,

 P < 0.01 vs control group;

 P < 0.05,

 P < 0.01 vs model group,

 P < 0.05,

 P < 0.01 vs G-CSF group “

” P < 0.05,

 P < 0.01 vs EPO group “

” P < 0.05,

P < 0.01 vs TPA12.5 μg · kg⁻¹ group

TABLE 4 The comparison of blood cell counts between each group (n = 10)(9 days after DNR + Ara-c, 1 day after 2^(nd) TPA or other) Group Dose(μg · kg⁻¹) WBC (×10⁹/L) RBC (×10¹²/L) PLT (×10⁹/L) Control —  6.72 ±0.81 9.74 ± 0.48 1432 ± 79.28 Model —  5.46 ± 0.49

5.45 ± 0.68

 512 ± 42.99

G-CSF 10  6.95 ± 0.44

5.40 ± 0.46

 949 ± 65.88

EPO 500  5.52 ± 0.34

6.75 ± 0.69

 935 ± 43.57

TPA 12.5  6.84 ± 0.44

7.47 ± 0.55

 965 ± 76.23

TPA 25  6.72 ± 0.50

7.34 ± 0.87

 951 ± 70.29

TPA 50  6.86 ± 0.39

7.30 ± 0.49

 999 ± 45.67

TPA + GCSF 12.5 + 10 10.74 ± 0.58

7.18 ± 0.38

 969 ± 52.86

TPA + EPO  12.5 + 500  6.78 ± 0.34

8.87 ± 0.29

 928 ± 32.72

Note: Date represent means SD.

 P < 0.05,

 P < 0.01 vs control group;

 P < 0.05,

 P < 0.01 vs model group,

 P < 0.05,

 P < 0.01 vs G-CSF group “

” P < 0.05,

 P < 0.01 vs EPO group “

” P < 0.05,

P < 0.01 vs TPA12.5 μg · kg⁻¹ group

TABLE 5 The comparison of blood cell counts between each group (n = 10)(10 days after DNR + Ara-c, 1 day after 3^(rd) TPA or other) Group Dose(μg · kg⁻¹) WBC (×10⁹/L) RBC (×10¹²/L) PLT (×10⁹/L) Control — 6.60 ±0.54  9.96 ± 0.82 1425 ± 52.34 Model — 6.55 ± 0.61  5.17 ± 0.56

 730 ± 71.09

G-CSF 10 9.14 ± 0.67

 5.30 ± 0.34

1456 ± 91.76

EPO 500 6.45 ± 0.41

 8.51 ± 0.86

1469 ± 75.67

TPA 12.5 9.51 ± 0.85

 7.59 ± 0.67

1447 ± 78.09

TPA 25 9.69 ± 0.85

 8.26 ± 0.63

1465 ± 72.81

TPA 50 9.90 ± 0.64

 8.18 ± 0.55

1463 ± 80.33

TPA + GCSF 12.5 + 10  9.90 ± 0.63

 7.47 ± 0.64

1419 ± 89.66

TPA + EPO 12.5 + 500 9.44 ± 0.30

10.18 ± 0.19

1430 ± 71.47

Note: Date represent means SD.

 P < 0.05,

 P < 0.01 vs control group;

 P < 0.05,

 P < 0.01 vs model group,

 P < 0.05,

 P < 0.01 vs G-CSF group “

” P < 0.05,

 P < 0.01 vs EPO group “

” P < 0.05,

P < 0.01 vs TPA12.5 μg · kg⁻¹ group

CONCLUSION

1) TPA promoted the production of WBC, RBC, and platelet. It could beuseful to treat different forms of cytopenia, such as anemia,leukopenia, neutropenia, thrombocytopenia, granulocypenia, pancytopenia,and hypocytopenia (see https at en.wikipedia.org/wiki/Cytopenia).2) TPA combined with G-CSF has a synergistic effect in promoting theproduction of WBC.3) TPA combined with EPO has a synergistic effect in promoting theproduction of RBC.4) TPA may promote the hematopoietic pathway on different stages, fromupstream myeloid stem cells to differentiate towards downstream stemcells and then from downstream stem cells to further differentiate todifferent blood cells.

I claim:
 1. A method of treating neutropenia and/or anemia comprisingadministering to a mammalian subject in need thereof, a combination of aphorbol ester of Formula I, pharmaceutically-acceptable salt, isomer,enantiomer, solvate, hydrate, or polymorph thereof,

wherein R₁ and R₂ are selected from the group consisting of hydrogen,hydroxyl,

wherein the alkyl group contains 1 to 15 carbon atoms,

wherein the lower alkenyl group contains 1 to 7 carbon atoms,

and

R₃ is hydrogen or

and a granulocyte-colony stimulating factor (G-CSF); wherein saidphorbol ester of Formula I is administered in an effective amount fromabout 10 and 1500 μg of said phorbol ester of Formula I per day, andsaid G-CSF is administered in an effective amount from 150 μg to 400 μg;wherein the G-CSF is administered to said mammalian subject in acoordinate administration protocol, simultaneously with, or afteradministration of said phorbol ester of Formula I; and wherein thecombination of phorbol ester of Formula I and G-CSF is administered atleast two times.
 2. A method of treating neutropenia and/orthrombocytopenia comprising administering to a mammalian subject in needthereof, a combination of a phorbol ester of Formula I,pharmaceutically-acceptable salt, isomer, enantiomer, solvate, hydrate,or polymorph thereof,

wherein R₁ and R₂ are selected from the group consisting of hydrogen,

wherein the alkyl group contains 1 to 15 carbon atoms,

wherein the lower alkenyl group contains 1 to 7 carbon atoms, and

R₃ is hydrogen or

and a granulocyte-colony stimulating factor (G-CSF); wherein saidphorbol ester of Formula I is administered in an effective amount fromabout 10 and 1500 μg of said phorbol ester of Formula I per day, andsaid G-CSF is administered in an effective amount from 150 μg to 400 μg;wherein the G-CSF is administered to said mammalian subject in acoordinate administration protocol, simultaneously with, or afteradministration of said phorbol ester of Formula I; and wherein thecombination of phorbol ester of Formula I and G-CSF is administered atleast two times.
 3. The method according to claim 1 or 2, wherein R₁ orR₂ is

the remaining R₁ or R₂ is

and R₃ is hydrogen.
 4. The method according to claim 1 or 2, wherein thephorbol ester is phorbol 13-butyrate, phorbol 12-decanoate, phorbol13-decanoate, phorbol 12,13-diacetate, phorbol 13,20-diacetate, phorbol12,13-dibenzoate, phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate,phorbol 12,13-dihexanoate, phorbol 12,13-dipropionate, phorbol12-myristate, phorbol 13-myristate, phorbol 12,13,20-triacetate,12-deoxyphorbol 13-angelate, 12-deoxyphorbol 13-angelate 20-acetate,12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate,12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol13-tetradecanoate, phorbol 12-tigliate 13-decanoate, 12-deoxyphorbol13-acetate, phorbol 12-acetate, or phorbol 13-acetate.
 5. The methodaccording to claim 1 or 2, wherein the phorbol ester is12-O-tetradecanoylphorbol-13-acetate (TPA).
 6. The method according toclaim 1 or 2, further comprising administering at least one secondary oradjunctive therapeutic agent.
 7. The method according to claim 1 or 2,wherein said phorbol ester of Formula I is administered every day orevery other day.
 8. The method according to claim 1 or 2, wherein saidphorbol ester of Formula I is administered in an effective amountcomprising between about 150 to 500 μg of said phorbol ester compound ofFormula I every day or every other day.
 9. The method according to claim1 or 2, wherein the combination increases absolute neutrophil count(ANC) of the mammalian subject to above 1500/mm³.
 10. The methodaccording to claim 1 or 2, wherein the combination increases plateletlevels of the mammalian subject to above 100,000/μl.
 11. The methodaccording to claim 1 or 2, wherein the mammalian subject is a human withacute myeloid leukemia (AML).